CN106596762A - Method for detecting content of atractylenolide III and alisol B 23-acetate in alisma decoction standard granules - Google Patents
Method for detecting content of atractylenolide III and alisol B 23-acetate in alisma decoction standard granules Download PDFInfo
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- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The present invention provides a method for detecting the content of atractylenolide III and alisol B 23-acetate in alisma decoction standard granules, the method can be used for achieving detection of the content of each component in each medicine in a compound traditional Chinese medicine preparation, namely simultaneous determination of the content of the atractylenolide III and the alisol B 23-acetate in the alisma decoction standard granules, and in particularly, under same chromatographic conditions, by use of a mobile phase gradient elution method, a DAD UV detector can be used for simultaneous determination of the content of the alisol B 23-acetate at 208nm and the content of the atractylenolide III at 222nm; and by methodology validation, the method is in full compliance with the specification, and is an advanced quality control method.
Description
(1) technical field
The present invention relates to a kind of content assaying method of Chinese medicine preparation, and in particular in one kind detection ZEXIE TANG standard particle
The method of atractylodes lactone III and Alisol B monoacetate content.
(2) background technology
ZEXIE TANG standard particle is the Prospect of TCM New Products that Zhengda Qingchunbao Pharmaceutical Co., Ltd develops, and prescription is derived from《Gold
Deficient outline》, it is made up of rhizoma alismatis and the taste medicine of the bighead atractylodes rhizome two.Fluid-retention in the epigastrium is cured mainly, lucid yang failing to raise, turbid YIN attacking upwards, the head is giddy, is now used for
Aural vertigo.
For the quality control of current compound Chinese medicinal preparation, each herbal medicine in can accomplishing for prescription has
One assay composition, is a kind of high quality standard of comparison.The present invention is for the rhizoma alismatis in ZEXIE TANG standard particle and in vain
Art this two herbal medicine, determines the content of Alisol B monoacetate in the content of atractylodes lactone III and rhizoma alismatis in the bighead atractylodes rhizome.By to this
The assay of two compositions, can better control over the quality of product and provide controllable, stable production technology.
(3) content of the invention
The present invention adopts high performance liquid chromatography, under same chromatographic condition, is eluted using eluent gradient, using DAD
UV-detector, atractylodes lactone III and Alisol B monoacetate in ZEXIE TANG standard particle are determined at the different wave length simultaneously
Content.
The technical solution used in the present invention is：
The method of atractylodes lactone III and Alisol B monoacetate content, described in a kind of detection ZEXIE TANG standard particle
Method is：
(1) testing sample is prepared
After by the finely ground mixing of ZEXIE TANG standard particle, with feed liquid mass volume ratio 1：5～20 (preferably 1：10～15, g：mL)
In adding methanol aqueous solution, ZEXIE TANG standard particle liquid is made, then in supersonic frequency 45KHz, 100～300W of ultrasonic power
Under the conditions of ultrasonically treated 10～30 minutes (preferably 20～30 minutes), be cooled to after room temperature add methanol aqueous solution supply weight extremely
With initial ZEXIE TANG standard particle liquid phase etc., shake up afterwards, filter (filter sizes are 0.45 μm), filtrate is taken, test sample is treated in acquisition
Product；
(2) testing sample spectrogram detection
Testing sample prepared by step (1) injects high performance liquid chromatograph and is detected, obtains the efficient of testing sample
Liquid chromatogram；The test condition of high performance liquid chromatograph is：Chromatographic column with octadecylsilane chemically bonded silica as filler, with
Distilled water is mobile phase A, and by Mobile phase B of acetonitrile gradient elution is carried out, and condition of gradient elution is：Mobile phase A during 0～10min
Volume fraction be 60%, during 10～50min the volume fraction of mobile phase A be 60%～10%, DAD UV-detectors it is same respectively
When at 208nm, 222nm detect absworption peak；
Preferably, the particle diameter of filler is 5 μm in the chromatographic column；The column length is 150mm, internal diameter is 4.6mm；
The flow velocity of eluant, eluent is 1.0ml/min during gradient elution；
During gradient elution, the volume fraction of the mobile phase A refers to that mobile phase A is total with Mobile phase B relative to mobile phase A
The volume fraction of volume；The volume fraction of mobile phase A is 60% during 0～10min, when referring to starting, the volume fraction of mobile phase A
For 60%, 0 to 10min is set, the volume fraction of mobile phase A keeps 60% constant；The volume integral of mobile phase A during 10～50min
Number is 60%～10%, refers to 10～50min, and the volume fraction of mobile phase A is from 60% linear decline to 10%；
(3) calibration curve of atractylodes lactone III and Alisol B monoacetate is made
Atractylodes lactone III reference substances methanol aqueous solution is dissolved, concentration is configured to for 5～50 μ g/ml (preferably 10～20
μ g/ml) atractylodes lactone III reference substance solutions, take atractylodes lactone III reference substance solutions and be injected separately into efficiently with different sample sizes
Liquid chromatogram is detected that the test condition of the high performance liquid chromatograph is identical with step (2), wherein DAD ultraviolet detections
Device detects absworption peak at 222nm, and the high performance liquid chromatography of the different atractylodes lactone III reference substance solutions of sample size is obtained respectively
Figure, the peak area of the absworption peaks of the atractylodes lactone III with high-efficient liquid phase chromatogram is molten with atractylodes lactone III reference substances as abscissa
Liquid sample size is ordinate, makes atractylodes lactone III calibration curves；
Atractylodes lactone III reference substances are replaced with into Alisol B monoacetate reference substance, DAD UV-detectors are at 208nm
Detection absworption peak, makes in the same way Alisol B monoacetate calibration curve；
(4) ZEXIE TANG standard particle assay
According to atractylodes lactone III in the high-efficient liquid phase chromatogram of step (2) gained testing sample at 222nm, 23- acetyl
The peak area at alisol B respective absorption peak at 208nm, and atractylodes lactone III, 23- acetylalisol that step (3) makes
The calibration curve of B, calculates the content of atractylodes lactone III and Alisol B monoacetate in testing sample, and then conversion obtains rhizoma alismatis
The content of atractylodes lactone III and Alisol B monoacetate in soup standard particle.
In detection method of the present invention, the volumetric concentration of methyl alcohol is 0～100% in used methanol aqueous solution, excellent
60% is selected, wherein " 0 " is meant that without methyl alcohol, it is only water.Also, the methanol aqueous solution being previously mentioned in step (1), (3) is equal
For the methanol aqueous solution of same volumetric concentration.
Heretofore described room temperature is 20～30 DEG C.
The advantage of the present invention is embodied in：Each herbal medicine in can accomplishing for compound Chinese medicinal preparation has a content
Composition is determined, i.e., while the content of the Alisol B monoacetate and atractylodes lactone III in measure ZEXIE TANG standard particle.Specially
Using same chromatographic condition, under the method for eluent gradient wash-out, determined at 208nm simultaneously using DAD UV-detectors
The content of Alisol B monoacetate, at 222nm determine atractylodes lactone III content.The content assaying method of the present invention is tested
Card complies fully with regulation, is a kind of advanced method of quality control.
There is the composition in the middle of composition and the bighead atractylodes rhizome in the middle of for rhizoma alismatis to carry out assay in published document at present
Report, but the composition in the middle of this two herbal medicine is not determined using same content assaying method simultaneously.And the present invention is right
This two herbal medicine of rhizoma alismatis and the bighead atractylodes rhizome in ZEXIE TANG standard particle is every kind of all to have done assay, and is in same chromatostrip
Measure under part.If detecting that the content of heterogeneity needs not with high performance liquid chromatography relative to general compound Chinese medicinal preparation
Same chromatographic condition, it is therefore desirable to substantial amounts of detection time and testing cost, can then save the time and detect into using the present invention
This, and can the more efficient quality for controlling product.
(4) illustrate
Fig. 1：Atractylodes lactone III canonical plottings in embodiment 1；
Fig. 2：Alisol B monoacetate canonical plotting in embodiment 1；
Fig. 3：The chromatogram of atractylodes lactone III reference substance solutions in embodiment 1, peak a is atractylodes lactone III；
Fig. 4：The chromatogram of Alisol B monoacetate reference substance solution in embodiment 1, peak b is Alisol B monoacetate；
Fig. 5：The atractylodes lactone III assay chromatograms of ZEXIE TANG standard particle, Detection wavelength 222nm in embodiment 1；
Fig. 6：The Alisol B monoacetate assay chromatogram of ZEXIE TANG standard particle, Detection wavelength in embodiment 1
208nm。
(5) specific embodiment
With reference to specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in
This.
Embodiment 1：
Using containing for atractylodes lactone III in high effective liquid chromatography for measuring ZEXIE TANG standard particle and Alisol B monoacetate
Amount.
(1) high-efficient liquid phase chromatogram condition
Instrument title：The high performance liquid chromatograph of Agilent 1260
Chromatographic condition：Chromatographic column：ZORBAX Eclipse Plus C18 (column length 150mm, internal diameter 4.6mm), filler：
Octadecylsilane chemically bonded silica (5 μm of particle diameter), flow velocity：1.0ml/min, column temperature：30℃
DAD UV-detector Detection wavelengths：208nm、222nm
With distilled water as mobile phase A, with acetonitrile as Mobile phase B, by table 1 gradient elution is carried out.
The condition of gradient elution of table 1 (% is percentage by volume)
| Time (minute) | Mobile phase A (%) | Mobile phase B (%) |
| 0～10 | 60 | 40 |
| 10～50 | 60→10 | 40→90 |
(2) preparation of reference substance solution：
A, atractylodes lactone III reference substance solutions：Precision weighs atractylodes lactone III reference substances, adds the first of volumetric concentration 60%
Alcohol solution, makes the atractylodes lactone III reference substance solutions of 12.29 μ g/ml.
B, Alisol B monoacetate reference substance solution：Precision weighs Alisol B monoacetate reference substance, adds volumetric concentration
60% methanol aqueous solution, makes the Alisol B monoacetate reference substance solution of 19.52 μ g/ml.
(3) preparation of testing sample solution：
ZEXIE TANG standard particle：Prescription is constituted：Rhizoma alismatis, the bighead atractylodes rhizome.Specification：4g/ bags.Producer：Honest QINGCHUN BAO medicine company is limited
Company.
Precision weighs the ZEXIE TANG standard particle 2g of finely ground mixing into conical flask with cover, adds the first of volumetric concentration 60%
Alcohol solution 25ml, precise weighing, ultrasonically treated 30 minutes, supersonic frequency 45khz, ultrasonic power 300w.Then take out, let cool
To room temperature.Weigh again, with the methanol aqueous solution of volumetric concentration 60% weight of less loss is supplied.Shake up, filter, take filtrate and obtain final product and treat
Survey sample solution.
(4) making of calibration curve：
The μ l of atractylodes lactone III reference substance solutions 2,4,6,8,12,16,20,30,40 injections for taking 12.29ng/ μ l respectively are high
Effect liquid phase chromatogram instrument, the test condition according to table 1 detected, absworption peak is detected at 222nm, with peak area as abscissa
(x), with atractylodes lactone III reference substance solution sample sizes (quality) as ordinate, (y) makes calibration curve, as a result as shown in Figure 1,
Y=0.425x+0.679 (R2=1).The high-efficient liquid phase chromatogram of atractylodes lactone III reference substance solutions is as shown in Figure 3.In the bighead atractylodes rhizome
Ester III linear relationships in the range of the sample introduction of 24.58ng～491.6ng are good.
The μ l of Alisol B monoacetate reference substance solution 2,4,6,8,12,16,20,30,40 notes of 19.52ng/ μ l are taken respectively
Enter high performance liquid chromatograph, the test condition according to table 1 is detected, absworption peak is detected at 208nm, with peak area as horizontal stroke
Coordinate (x), with Alisol B monoacetate reference substance solution sample size (quality) as ordinate, (y) makes calibration curve, as a result sees
Shown in Fig. 2, y=0.872x-5.331 (R2=1).The high-efficient liquid phase chromatogram of Alisol B monoacetate reference substance solution is shown in Fig. 4
It is shown.Alisol B monoacetate linear relationship in the range of the sample introduction of 39.04ng～780.8ng is good.
(5) testing sample assay：
The μ l of testing sample solution 10 that accurate aspiration step (3) is prepared, inject high performance liquid chromatograph, according to table 1
Test condition detected, absworption peak is detected simultaneously at 208nm and 222nm respectively, according to atractylodes lactone III in testing sample
At 222nm, the peak area at Alisol B monoacetate respective absorption peak at 208nm, and atractylodes lactone III calibration curves and
Alisol B monoacetate calibration curve, calculates atractylodes lactone III and Alisol B monoacetate content in testing sample, and then calculates
Obtain atractylodes lactone III and Alisol B monoacetate content in ZEXIE TANG standard particle.
As a result it is：In per bag of (4g) ZEXIE TANG standard particle atractylodes lactone III contents be 0.597mg, 23- acetylalisols
B content is 1.671mg.Shown in high-efficient liquid phase chromatogram as Fig. 5 and Fig. 6, Fig. 5 treats test sample for Detection wavelength measure at 222nm
The high-efficient liquid phase chromatogram of the atractylodes lactone III in product solution, Fig. 6 determines testing sample solution for Detection wavelength at 208nm
In Alisol B monoacetate high-efficient liquid phase chromatogram.
Embodiment 2：
Take with a batch of ZEXIE TANG standard particle, carry out content assaying method checking, experimental implementation with embodiment 1,
Obtain testing sample solution.
Precision investigation (repeated experiment, Intermediate precision experiment) is carried out, the results are shown in Table shown in 2,3.As a result show,
The RSD of assay meets requirements of the RSD within 3% within 2%.
Degree of accuracy investigation (rate of recovery experiment) is carried out, the results are shown in Table shown in 4.As a result meet the rate of recovery 95%～
105% area requirement, RSD meets the requirement within 5%.
24 hours study on the stability of testing sample solution are carried out, the results are shown in Table shown in 5.As a result meet RSD 2% with
Interior requirement.
The inventive method repeated experiment of table 2
The inventive method Intermediate precision of table 3 is tested
The inventive method degree of accuracy of table 4 is tested
The inventive method Stability Determination of table 5
| 0h | 2h | 4h | 8h | 16h | 20h | 24h | Peak area RSD% | |
| Atractylodes lactone III peak areas | 275.2 | 275.3 | 275.2 | 275.5 | 276.1 | 276.7 | 276 | 0.43 |
| Alisol B monoacetate peak area | 363.7 | 361.9 | 359.8 | 361 | 361.9 | 360.5 | 359.6 | 0.77 |
Claims (8)
1. in a kind of detection ZEXIE TANG standard particle atractylodes lactone III and Alisol B monoacetate content method, its feature exists
In described method is：
(1) testing sample is prepared
After by the finely ground mixing of ZEXIE TANG standard particle, with feed liquid mass volume ratio 1：5～20 add in methanol aqueous solution, make pool
Soup standard particle liquid is rushed down, then ultrasonically treated 10～30 points under conditions of supersonic frequency 45KHz, 100～300W of ultrasonic power
Clock, be cooled to after room temperature add methanol aqueous solution supply weight to initial ZEXIE TANG standard particle liquid phase etc., shaking up afterwards, mistake
Filter, takes filtrate, obtains testing sample；
(2) testing sample spectrogram detection
Testing sample prepared by step (1) injects high performance liquid chromatograph and is detected, obtains the efficient liquid phase of testing sample
Chromatogram；The test condition of high performance liquid chromatograph is：Chromatographic column with octadecylsilane chemically bonded silica as filler, with distill
Water is mobile phase A, and by Mobile phase B of acetonitrile gradient elution is carried out, and condition of gradient elution is：The body of mobile phase A during 0～10min
Fraction is 60%, and the volume fraction of mobile phase A is that 60%～10%, DAD UV-detectors exist respectively simultaneously during 10～50min
Absworption peak is detected at 208nm, 222nm；
(3) calibration curve of atractylodes lactone III and Alisol B monoacetate is made
Atractylodes lactone III reference substances methanol aqueous solution is dissolved, concentration is configured to for 5～50 μ g/ml atractylodes lactones III controls
Product solution, takes atractylodes lactone III reference substance solutions and is injected separately into high performance liquid chromatography with different sample sizes and detected, described
The test condition of high performance liquid chromatograph is identical with step (2), and wherein DAD UV-detectors detect absworption peak at 222nm,
The high-efficient liquid phase chromatogram of the different atractylodes lactone III reference substance solutions of sample size is obtained respectively, with high-efficient liquid phase chromatogram
The peak area of atractylodes lactone III absworption peaks is abscissa, with atractylodes lactone III reference substance solution sample sizes as ordinate, is made
Atractylodes lactone III calibration curves；
Atractylodes lactone III reference substances are replaced with into Alisol B monoacetate reference substance, DAD UV-detectors are detected at 208nm
Absworption peak, makes in the same way Alisol B monoacetate calibration curve；
(4) ZEXIE TANG standard particle assay
According to atractylodes lactone III in the high-efficient liquid phase chromatogram of step (2) gained testing sample at 222nm, 23- acetyl rhizoma alismatis
The peak area at alcohol B respective absorption peaks at 208nm, and atractylodes lactone III, the Alisol B monoacetate that step (3) makes
Calibration curve, calculates the content of atractylodes lactone III and Alisol B monoacetate in testing sample, and then conversion obtains ZEXIE TANG mark
The content of atractylodes lactone III and Alisol B monoacetate in quasi- particle.
2. atractylodes lactone III and Alisol B monoacetate content in detection ZEXIE TANG standard particle as claimed in claim 1
Method, it is characterised in that the volumetric concentration of methyl alcohol is 0～100% in the methanol aqueous solution.
3. atractylodes lactone III and Alisol B monoacetate content in detection ZEXIE TANG standard particle as claimed in claim 1
Method, it is characterised in that the volumetric concentration of methyl alcohol is 60% in the methanol aqueous solution.
4. atractylodes lactone III and Alisol B monoacetate content in detection ZEXIE TANG standard particle as claimed in claim 1
Method, it is characterised in that in step (1), after the described finely ground mixing of ZEXIE TANG standard particle, with feed liquid mass volume ratio 1：10
～15 add in methanol aqueous solution.
5. atractylodes lactone III and Alisol B monoacetate content in detection ZEXIE TANG standard particle as claimed in claim 1
Method, it is characterised in that in step (2), the particle diameter of filler is 5 μm in the chromatographic column.
6. atractylodes lactone III and Alisol B monoacetate content in detection ZEXIE TANG standard particle as claimed in claim 1
Method, it is characterised in that in step (2), the column length is 150mm, internal diameter is 4.6mm.
7. atractylodes lactone III and Alisol B monoacetate content in detection ZEXIE TANG standard particle as claimed in claim 1
Method, it is characterised in that in step (2), the flow velocity of eluant, eluent is 1.0ml/min during gradient elution.
8. atractylodes lactone III and Alisol B monoacetate content in detection ZEXIE TANG standard particle as claimed in claim 1
Method, it is characterised in that in step (3), the concentration of the atractylodes lactone III reference substance solutions is 10～20 μ g/ml.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110441407A (en) * | 2018-05-03 | 2019-11-12 | 天津药物研究院有限公司 | A kind of pool art tablet quality control method |
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| CN112213409A (en) * | 2020-05-19 | 2021-01-12 | 青海普兰特药业有限公司 | Detection method of UPLC characteristic spectrum of Alismatis rhizoma decoction and application of characteristic spectrum |
| CN116754665A (en) * | 2023-05-19 | 2023-09-15 | 广东一方制药有限公司 | Detection method of Alisma decoction |
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